RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small RNA molecules—micro RNA (miRNA) and small interfering RNA (siRNA)—are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA (mRNA) from producing a protein. RNA interference has an important role in defending cells against parasitic genes but also in directing development as well as gene expression in general.
The RNAi pathway is found in many eukaryotes and is initiated by the enzyme Dicer which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ˜20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing comples (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
RNA interference (RNAi) is a post-transcriptional gene-silencing mechanism, by which a specific degradation of mRNA is induced by homologous double-stranded RNAs (dsRNAs), and constitutes a powerful tool for gene function analysis.
RNAi can occur by introducing chemically synthesized siRNA or by endogenous microRNAs (miRNA) that silence cellular mRNAs (miRNAs). Reviewed in Sledz C A, Williams B R. 2005. RNA interference in biology and disease. Blood. 106(3):787-94).
The synthetic dsRNA of 21-23 nucleotides that target a specific region in the mRNA is a synthetic siRNA. Taken advantage of miRNA structure, short hairpin forms (e.g., expressed from shRNA vectors) are about 50-70 nucleotides that comprise sense strand, loop sequences, antisense strand and termination sequence. Once in cells, the hairpins are processed by dicer enzyme into an siRNA that mediates gene silencing. In mammalian cells, the antisense strand of synthetic short interfering RNA (siRNA) serves as a template for the RNA-induced silencing complex (RISC) to recognize and cleave complementary messenger RNA (mRNA), which is then rapidly degraded.
The short inhibitory RNAs (siRNAs) are commonly made as chemically synthesized double-stranded short RNA (19-29 mer) which is relatively easy and has a higher transfection rate. However, it is costly and transient in action. Vector based RNAi approaches included the use of short hairpin RNA (shRNA) as plasmid or virus-based vectors.
The need for inducible (regulated) RNAi vectors allow the precise and temporal control of gene knockdown, to avoid artifactual differences between compared cell populations, reduction of non-specific cellular effects due to induction of transient expression, and for expression of RNAi when extended can cause cellular toxicity or unwanted changes.
The problem is that once RNAi vectors are made, inclusion of regulated sequences, such as tetracycline operator (TetO) sites, require time consuming cloning and use of enzymes, such as restriction enzymes, ligase, topoisomerase, or recombinase. Though the TetO-sites can be synthesized as double stranded polynucleotides or as a PCR product, these still need to be cloned into the vector using restriction sites and cloning enzymes. All of these cloning-based techniques require propagation of the plasmid (including transformation into bacterial cells, culture growth, and plasmid extraction) or the virus-based vector include packaging in cells and virus purification. These cloning-based processes become almost formidable when working with large number of RNAi vectors such as in high-throughput applications.
Thus, there is a need in the art to improve the methods of providing RNAi sequences in an expressable (ready-to-use) format.
There is furthermore a need in the art to improve the methods of providing a (any) gene sequence of interest in an expressable (ready-to-use) format that is simple to handle and/or which furthermore allows assessing post-transcriptional effects.